Osteocytes are critical to the maintenance of tissue quality and mechanical integrity of bone. As the primary mechanosensing cells, osteocytes orchestrate bone's adaptation processes under mechanical cues such as load-induced fluid flow. However, the in vivo mechanisms by which osteocytes, deeply embedded in mineralized matrix, detect and transduce mechanical signals remain elusive. Filling this knowledge gap is essential to the development of new osteoporosis treatments that exploit bone's intrinsic sensitivity to mechanical loading (a potent anabolic factor). Recent studies have found a fibrous pericellular matrix (PCM) that spans the entire fluid annulus (~80nm) within the lacunar-canalicular system (LCS) and tethers the cell processes to the canalicular wall matrix. Evidence increasingly suggests that these PCM tethering fibers act as mechanical sensors, capturing fluid drag force and initiating mechanotransduction cascades in osteocytes. However, rigorous testing of this concept has been hindered by a lack of quantitative tools for measuring the PCM ultrastructure and by the scarcity of data regarding PCM composition. Breakthroughs from our previous award cycle have overcome these barriers, allowing us to precisely define the functional roles of the PCM in bone. First, we invented a tracer velocimetry approach based on fluorescence recovery after photobleaching (FRAP) to quantify osteocytic PCM in intact bone. Second, we identified perlecan/HSPG2, a large heparan sulfate (HS) proteoglycan, to be an essential structural component of the PCM. Using a perlecan-deficient mouse model that mimics human Schwartz-Jampel syndrome (SJS) we discovered that perlecan deficiency results in not only decreased PCM fiber density but also attenuated responses to in vivo loading and unloading. These preliminary studies formed the cornerstone of our hypothesis that the osteocytic PCM regulates bone's adaptation to mechanical cues through mechanosensing in the LCS, which will be tested at the tissue, cellular, and molecular levels in the following three specific aims: 1) Quantify the effects of PCM alterations on bone adaptation to mechanical cues in vivo; 2) Quantify the effects of PCM alterations on osteocyte mechanosensing ex vivo; 3) Determine the mechanisms by which PCM perlecan forms functional mechanosensing tethers in the LCS in vitro. The proposed studies are important because PCM is the critical interface between osteocytes and the extracellular environment. Identifying the functional roles of the osteocytic PCM and one of its major components, perlecan, in bone adaptation could lead to the development of new osteoporosis treatments that exploit bone's intrinsic sensitivity to mechanical stimuli, a potent non- pharmaceutical factor in promoting bone formation. These studies will also advance our knowledge of the fundamental functions of the PCM, a uniquely functioning but overlooked structure found in nearly all mammalian cells including osteocytes.